A number of important neurological diseases including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and prion diseases are characterized by the deposition of aggregated proteins, referred to as amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al. (1989) J. Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310. These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of a .beta.-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). AD studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common cause of progressive intellectual failure in aged humans, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review see Terry et al. (1994) "Structural alteration in Alzheimer's disease." In: Alzheimer's disease (Terry et al. eds.), pp. 179-196. Raven Press, New York). Dystrophic neurites, as well as reactive astrocytes and microglia, are embedded in the core of these amyloid-associated neurite plaques. Although, the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy (1997) Trends Neurosci. 20:154-9; Haan et al. (1990) Clin. Neurol. Neurosurg. 22:305-10; Terry et al., supra; Vinters (1987) Stroke 18:211-24; Itoh et al. (1993) J. Neurological Sci. 116:135-41; Yamada et al. (1993) J. Neurol. Neurosurg. Psychiatry 5:543-7; Greenberg et al. (1993) Neurology 43:2073-9; Levy et al. (1990) Science 2:1124-6). In some familial CAA cases, dementia was noted before the onset of hemorrhages (27), suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
In both AD and CAA, the main amyloid component is the amyloid .beta. protein (A.beta.). The A.beta. peptide, which is generated from the amyloid .beta. precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A.beta..sub.1-40 and A.beta..sub.1-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). A.beta..sub.1-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well-defined. However, evidence indicates that disregulated expression and/or processing of APP gene products or derivatives of these gene products derivatives are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy (1994) Clin. Geriatr. Med. 10:239-247; Mann et al. (1992) Neurodegeneration 1:201-215). The role of APP in neurodegenerative disease is further implicated by the observation that persons with Down's syndrome who carry an additional copy of the human APP (hAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. (1993) J. Neuropathol. Exp. Neurol. 52:192-198; Oyama et al. (1994) J. Neurochem. 62:1062-1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. (1985) Ann. Neurol. 17:278-282). Mutations in A.beta. are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch (HCHWA-D)(Levy et al. (1990), supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. (1994) Acta Neuropathol. 88:371-8; Wattendorff et al. (1995) J. Neurol. Neurosurg. Psychiatry 5:699-705). A number hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the A.beta. peptide (for a review, see, e.g., Lannfelt et al. (1994) Biochem. Soc Trans. 22:176-179; Clark et al. (1993) Arch. Neurol. 50:1164-1172). Finally, in vitro studies indicate that aggregated A.beta. can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253-265).
APP is encoded by a 19-exon gene: exons 1-13, exon 13a, and 14-18 (Yoshikai et al. (1990) Gene 82:257-263; see FIG. 1 for a map of the hAPP exon-intron organization of the hAPP gene). Alternative splicing of APP gene-derived transcripts results in at least 10 isoforms (Sandbrink et al. (1994) J. Biol. Chem. 269:1510-1517). The predominant transcripts are APP695 (exons 1-6, 9-18, not 13a), APP751 (exons 1-7, 9-18, not 13a) and APP770 (exons 1-18, not 13a). All of these encode multidomain proteins with a single membrane-spanning region. They differ in that APP751 and APP770 contain exon 7, which encodes a serine protease inhibitor domain. APP695 is a predominant form in neuronal tissue, whereas APP751 is the predominant variant elsewhere. A.beta. amyloid is derived from that part of the protein encoded by parts of exons 16 and 17.
In three APP mutants, valine-642 in the transmembrane domain of APP(695) is replaced by isoleucine, phenylalanine, or glycine in association with dominantly inherited familial Alzheimer disease. (According to an earlier numbering system, val642 was numbered 717 and the 3 mutations were V717I, V717F, and V717G, respectively.) Yamatsuji et al. ((1996) Science 272:1349-1352) concluded that these three mutations account for most, if not all, of the chromosome 21-linked Alzheimer disease. Suzuki et al. ((1994) Science 264:1336-1340) suggested that these mutations may cause Alzheimer disease by altering .beta.-APP processing in a way that is amyloidogenic. They found that the APP-717 mutations were consistently associated with a 1.5- to 1.9-fold increase in the percentage of longer A.beta. fragments generated and that the longer fragments formed insoluble amyloid fibrils more rapidly than did the shorter ones. In transgenic mice, overexpression of such mutants mimics the neuropathology of AD.
Several transgenic animals expressing human APP (hAPP) have been developed as models for AD (Higgins et al. (1994) Ann. Neurol 35:598-607; Mucke et al. (1994) Brain Res. 666:151-167; Games et al. (1995) Nature 373:523-527; Games et al. (1995) Soc. Neurosci Abstr. 21:258). Most transgenic models were designed based on the observation that a number of APP mutations cosegregate with the familial form of AD; patients carrying these APP mutations exhibit neuropathological alterations that are indistinguishable from sporadic AD (Chartier-Harlin et al. (1991) Nature 353:844-846; Goate et al. (1991) Nature 349:704; Murrell et al. (1991) Science 254:97-99; Clark et al. (1993) Arch. Neurol. 50:1164-1172). Moreover, APP717 mutations result in an overproduction of the highly amyloidogenic A.beta..sub.1-42 relative to other A.beta. peptides (Suzuki et al. (1994) Science 264:1336-1340).
At least one of these transgenic animal models exhibits AD-like neuropathology (Games et al. (1995) Nature 373:523-527; Masliah et al. (1996) J. Neurosci. 16:5795-5811). The transgenic mouse of Games et al. carries an hAPP minigene (PDAPP), where expression of the PDAPP minigene is driven by a platelet-derived growth factor-B chain (PDGF-B) promoter (Games et al (1995) Nature 373:523-527; Rockenstein et al. (1995) J. Biol. Chem. 270:28257-28267). The hAPP minigene encodes an alternatively spliced hAPP containing the mutation V.fwdarw.F (valine to phenylalanine) at residue 717 (APP.sub.717V.fwdarw.F), a mutation associated with familial AD (Chartier-Harlin et al. (1991), supra; Murrell et al. (1991), supra; Clark et al. (1993), supra). The PDAPP minigene contains three modified hAPP introns that differed from the corresponding authentic hAPP gene introns by large deletions (introns 6 and 8) or insertion of four nucleotides (intron 7) (Rockenstein et al. (1995), supra). The PDAPP transgenics exhibited four to six-fold higher levels of total APP mRNA relative to nontransgenic animals. In addition, the transgenics' endogenous APP mRNA levels were reduced, resulting in a high ratio of mRNA encoding mutated hAPP versus wild-type murine APP.
The PDAPP transgenic animal of Games et al. ((1995), supra) exhibited both age- and brain region-dependent development of typical amyloid plaques, dystrophic neurites, loss of presynaptic terminals, astrocytosis, and microgliosis. The brains showed typical pathologic findings of AD, including numerous extracellular thioflavin S-positive A-beta deposits, neuritic plaques, synaptic loss, astrocytosis, and microgliosis. A.beta. deposits were observed primarily in the hippocampus and cerebral cortex; moreover, A.beta. deposits increased with the animal's age.
Hsiao et al. ((1996) Science 274:99-102) produced transgenic mice overexpressing the 695-amino acid isoform of human APP containing a K670N, M671L double mutation, which was described by Mullan et al. ((1992) Nature Genet. 1:345-349) in a large Swedish family with early-onset Alzheimer disease. Hsiao et al. ((1996) Science 274:99-102) reported that a 5-fold increase in the concentration of the .beta. amyloid derivatives was found in the brains of the older transgenic mice; older mice showed impairment in both learning and memory in spatial reference and alternation tasks. Classic senile plaques with dense amyloid cores were present in mice with elevated brain beta amyloid.
Although presently available transgenic animals are promising model for AD and AD-related neuropathologies, the amount of time required to detect AD-like pathology is quite long. For example, the transgenic mice of Games et al. ((1995), supra) exhibited no obvious pathology between four to six months of age. Although Games et al.'s transgenics began to exhibit deposits of human A.beta. at about six to nine months, with many deposits observable by eight months, it was not until the transgenic animals were about nine months old or older that the density of plaques increased so that the A.beta.-staining pattern resembled that of AD (Games et al., (1995), supra). Likewise, the transgenic animals of Hsaio et al. had normal learning and memory in spatial reference and alternation tasks at 3 months of age; impairment in these characteristics were not apparent until the transgenics were 9 to 10 months of age.
The development of animal models for AD and CAA is a critical step for both understanding these diseases and developing therapeutic drugs. However, the present AD animal models are less desirable, at least in part, because the transgenic animals must be maintained for nearly a year before AD-like pathology is observable, thus significantly slowing the ability to assess the prophylactic or therapeutic effects of candidate drugs for AD and/or AD-like conditions. Worse still, there are no suitable models to study CAA. Thus, there is a clear need in the field for an animal model of AD and CAA.